Identification and characterization of novel lineage 1 Powassan virus strains in New York State

Figures & data

Table 1. Recent and historic POWV-1 and DTV isolates collected in New York State.

Strain	County	Year	Host	Lineage
POWV-1 64–7062	St. Lawrence	1964	I. cookei	1
POWV-1 46–001	Jefferson	2018	I. cookei	1
POWV-1 63–002	Orange	2018	I. cookei	1
POWV-1 19065–059	Oneida	2019	I. scapularis	1
DTV 18071–054	Orange	2018	I. scapularis	2

Figure 1. Powassan virus (POWV-1) and deer tick virus (DTV) surveillance across New York State (NYS) between 2007-2019. All counties are sampled yearly for questing adult Ixodes scapularis and tested for POWV except for 4 counties outside of New York City. Counties shaded in gray represent those in which at least 1 tick has been positive for POWV and those in white represent no viral detection. Counties with stars indicate regions which recent NYS POWV-1 isolates were collected. Stars do not represent location within the county of the collection site and were placed concentrically.

Figure 2. Maximum likelihood phylogeny of Powassan virus (POWV-1) and deer tick virus (DTV) based on full genome nucleotide sequences with tickborne encephalitis virus as an outgroup. Bootstrap values are displayed at each node (range: 38.9-100). Recent New York State (NYS) isolates (arrows); POWV-1 46-001, POWV-1 63-002, and POWV-1 19065–059 cluster with historic POWV-1 isolates.

Table 2. Amino acid substitutions for recent Powassan virus (POWV-1) isolates from New York State (NYS) as compared to all available sequenced POWV-1 and deer tick virus (DTV) isolates. Unique substitutions are bolded. Lineage-2-like substitutions are highlighted in light grey.

Genome Position	AA Position	POWV-1 46–001	POWV-1 63–002	POWV-1 19065–059	POWV-1 64–7062	DTV 18071–054
PrM	259	GLY	SER	SER	SER	SER
PrM	262	VAL	ALA	ALA	ALA	ALA
PrM	265	THR	THR	ILE	THR	THR
E	312	THR	THR	SER	THR	THR
E	366	PRO	ALA	ALA	ALA	ALA
E	404	ARG	LYS	LYS	LYS	LYS
E	405	ALA	VAL	ALA	ALA	ALA
E	421	ALA	VAL	VAL	VAL	VAL
E	439	LYS	LYS	ARG	LYS	LYS
E	453	ARG	ARG	GLN	ARG	ASN
E	687	ARG	LYS	ARG	ARG	LYS
NS1	1004	ALA	PRO	ALA	ALA	ALA
NS2a	1292	ARG	ARG	LYS	ARG	ARG
NS2a	1303	VAL	ILE	VAL	VAL	VAL
NS2a	1339	SER	PRO	SER	SER	PRO
NS2b	1416	VAL	ALA	VAL	VAL	ALA
NS3	1660	SER	ASN	ASN	ASN	SER
NS3	1930	ILE	VAL	ILE	ILE	ILE
NS4a	2121	SER	GLY	GLY	GLY	GLY
NS4b	2286	VAL	ALA	ALA	VAL	VAL
NS4b	2315	MET	VAL	MET	MET	MET
NS4b	2415	ILE	ILE	VAL	ILE	ILE
NS5	2538	ALA	ALA	SER	ALA	ALA
NS5	2692	VAL	ALA	ALA	ALA	ALA
NS5	3018	ALA	ALA	SER	ALA	ALA

Figure 3. Growth kinetics of Powassan virus (POWV-1) and deer tick virus (DTV) in baby hamster kidney (BHK-21) cells. Replication in BHK-21 is similar among all isolates. Data points represent mean +/- SEM (n = 3 per strain). Two-way ANOVA and Tukey’s multiple comparisons test (p = 0.8261). 

Figure 4. Overall infection rates of New York State (NYS) Powassan virus (POWV-1) and deer tick virus (DTV) isolates in Ixodes scapularis nymphs following immersion. Black shading represents the percent of infected ticks with POWV-1 or DTV and gray shading represent the percent of uninfected ticks. Infected ticks across all timepoints (7-,14-,21-, and 28-days post infection) were combined to calculate overall percentage of POWV-infected ticks (n = 80/strain for POWV-1 64–7062 and POWV-1 46-001, n = 90/strain for POWV-1 63-002, POWV-1 19065-059, and DTV 18071-054). POWV-1 64-7062, POWV-1 46-001, and POWV-1 63–002 displayed significantly lower infection rates compared to POWV-1 19065–059 and DTV 18071–054 (chi-squared test, **p < 0.01, ***p < 0.001).

Figure 5. Growth kinetics of New York State (NYS) Powassan virus (POWV-1) and deer tick virus (DTV) isolates in Ixodes scapularis nymphs following immersion. Individual data include two separate, statistically equivalent experiments (t-test, p > 0.05, n = 4-19). Time (DPI) significantly influenced viral load (two-way ANOVA, p < 0.0001). Pairwise comparisons indicated statistical equivalence of viral load among strains, with the exception of POWV-1 63-002, which was significantly lower than all other strains at 21 DPI (two-way ANOVA, Tukey’s multiple comparisons, p < 0.017).